Kit for the determination of l-carnitine in biological fluids and tissues

ABSTRACT

A kit is described for the determination of L-carnitine in biological fluids and tissues, consisting essentially of; a) a first container containing Reagent 1, said Reagent 1 comprising HEPES, EDTA, acetyl-coenzyme A and DTNB; b) a second container containing Reagent 2, said Reagent 2 consisting of carnitine acetyl transferase. Said kit is conveniently presented with the reagents in lyophilized form.

[0001] The invention described herein relates to a kit for thedetermination of L-carnitine in biological fluids.

BACKGROUND TO THE INVENTION

[0002] L-carnitine plays a fundamental role in metabolism, being a keyelement in the oxidation of long-chain fatty acids and thus in theproduction of energy.

[0003] There are many pathological states the underlying cause of whichis a deficiency of L-carnitine, and L-carnitine determination isnecessary in order to establish the precise aetiology of the relateddisease processes.

[0004] There has been a growing awareness among clinicians over recentyears of the importance of L-carnitine deficiencies (Bieber et is al.Fed. Proc. 41, 2858 (1982); Stanley, Adv. Pediatr. 34, 59 (1987).

[0005] One of the first methods used for determining L-carnitine(hereinafter called carnitine for short) was described by Marquis andFritz in 1964 (Journal of Lipid Research 5, 184-187). Various othermethods have been described, such as, for example, those by Marzo etal., J. Chromatogr. 527, 247 (1990); and Hoppel, in: Hommes (ed.),Techniques in Diagnostic Human Biochemical Genetics, New York,Wiley-Liss, 309-326 (1991).

[0006] The most widespread methods are based on the reaction of theenzyme carnitine acetyl transferase (CAT; EC 2.3.1.7):

[0007] One very widespread method for the indirect determination ofcarnitine is based on the reaction of acetyl-Coenzyme A, released by thepreceding reaction with 5,5′-dithiobis-2-nitrobenzoate (DTNB), which inturn releases the thiophenylate ion, which is determinedspectrophotometrically at 412 nm (see, in addition to theabove-mentioned Marquis, Seccombe D. W., Clinical Chemistry, 22, No. 10,1589-1592, 1976; Pearson, Methods of Enzymatic Analysis, 4, 1758-1771,2nd edition—Bergmeyer; Casillas E. R. Biochimica et Biophysica Acta,184, 566-577, 1969; Cederblad G. Clinica Chimica Acta, 33, 117.123,1971; Carrier H. N., Muscle & Nerve July/August 326-328, 1980). Thismethod determines free carnitine. For the determination of theshort-chain acyl carnitines, and thus of total carnitines, the sample issubjected to alkaline hydrolysis, converting the acyl carnitines to freecarnitine.

[0008] The known methods entail elaborate preparation phases, or usetechniques which are only poorly conducive to automation of theanalyses. For a discussion of the subject, see U.S. Pat. No. 5,316,917,filed in the name of Duke University, and incorporated herein forreference purposes. This patent aims to solve the problem of theautomation of the analyses. This need is strongly felt in clinicallaboratories that have to carry out large numbers of determinations. TheDuke University patent offers the solution of an automatedspectrophotometric method, consisting in the following steps:

[0009] 1) addition of a plurality of samples of deproteinized biologicalfluids to a set of wells of a centrifugal spectrophotometric analyzer;

[0010] 2) addition to each sample of:

[0011] a) acetyl-Coenzyme A in an amount sufficient to react essentiallywith all the free carnitine in the sample to produce acetyl-carnitineand free Coenzyme A;

[0012] b) DTNB in an amount sufficient to convert essentially all theCoenzyme A produced in the previous reaction to thiophenylate, and then

[0013] c) simultaneously combine CAT, in each sample, in an amountsufficient to set off the reaction between acetyl-Coenzyme A and freecarnitine and bring it to completion, and then

[0014] d) determine the amount of thiophenylate present in the samplesimultaneously by spectrophotometry.

[0015] The kit used to implement the method described in the patentcited includes:

[0016] a) a first container containing a solution of DTNB at aconcentration ranging from 0.27 to 27 mmol/L, at a pH ranging from 6.5to 8.5, and

[0017] b) a second container containing a solution consisting ofacetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L,where the two solutions can be mixed together prior to use to form asolution containing DTNB at a concentration ranging from 0.23 to 23mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17mmol/l.

[0018] In actual fact, the kit must also include a third containercontaining from 4 to 40 kU/L of CAT, for example in the form of asolution at a concentration ranging from 1.72 to 172 kU/L. It isenvisaged that the third container should contain CAT in lyophilizedform. Also envisaged in the kit are containers with carnitine standardsin aqueous solution from 0.1 to 10 mmol/L and of octanoyl L-carnitinefrom 0.1 to 10 mmol/L. Also provided in the kit is a fourth containercontaining a 3-[N-morpholine]propanesulphonic acid hydrochloricsolution, from 0.1 to 10 mmol/L.

[0019] The execution of the procedure entails the preparation of 4solutions:

[0020] 1. DTNB; HEPES; EDTA at pH 7.5

[0021] 2. MOPS-HCl

[0022] 3. KOH

[0023] In fact, the procedure described in the patent is called “threereagent chemistry”.

[0024] It is known that the solutions envisaged in the kit describedabove must be stored at low temperature, from 0 to 4° C., as prescribedin the patent cited.

[0025] U.S. Pat. No. 6,027,690, a divisional patent of U.S. Pat. No.5,872,008, filed in the name of Bair and Shug, provides a kit for thediagnosis of premenstrual syndrome, based on the determination of freeand total carnitine in the blood. Apart from the specific indication ofthe method, to implement the method the kit consists of:

[0026] a) a first container containing a solution of acetyl-Coenzyme Aat a concentration ranging from 1.2 to 120 mmol/L, and

[0027] b) a second container containing a solution consisting of DTNB orN-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from0.27 to 27 mmol/L at a pH ranging from 6.5 to 8.5, where the twosolutions can be mixed together prior to use to form a solutionconsisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at aconcentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at aconcentration ranging from 0.17 to 17 mmol/l.

[0028] In the execution of the procedure according to U.S. Pat. No.6,072,690, the method described in U.S. Pat. No. 5,316,917 can beadopted amongst others.

[0029] A Boehringer Mannheim kit is commercially available for thedetermination of L-carnitine in plasma, seminal fluid, and urine. Thedetermination is based on the following reactions:

[0030] The amount of NADH consumed during the reaction is equivalent tohalf the amount of L-carnitine. NADH is determined by absorbance at 334(Hg), 340 or 365 (Hg) nm.

[0031] The kit comprises:

[0032] 1) 3 vials (1) each containing 0.7 g of a Coenzyme/buffer mixtureconsisting of Tris buffer pH 7.0, NADH 5 mg, ATP 6 mg, acetyl-Coenzyme A4 mg, PEP 3 mg, magnesium acetate, and stabilizing agents.

[0033] 2) Vial (2) containing approximately 3 ml of enzyme suspensionconsisting of acetyl-CoA synthetase (ACS), 2U approx., myokinase (MK),160 U approx., lactate dehydrogenase (LDH), 240 U approx., and pyruvatekinase (PK), 240 U approx.

[0034] 3) Vial (3) containing 0.2 ml of carnitine acetyl transferase(CAT) enzyme suspension, 60 U approx.

[0035] 4) Vial (4) containing L-carnitine standard.

[0036] 5) Vial (5) containing detergent solution.

[0037] Prior to use, the contents of one vial (1) are diluted with 10 mlof distilled water and 1 ml is added from vial (5). The other vials areused as such. The procedure consists in the addition of the sample (orstandard) to solution (1), then addition of suspension (2), measurementof absorbance, then addition of suspension (3) and subsequentmeasurement of absorbance.

ABSTRACT OF THE INVENTION

[0038] It has now been found that it is possible to provide a kit forthe determination of L-carnitine or short-chain acyl L-carnitinesconsisting of only two vials complete with all the necessary reactants,with the exception of the standards. Conveniently, the kits contain thereactants in lyophilized form.

[0039] One of the advantages afforded by the realization of theinvention described herein consists in the possibility of determiningcarnitine over a broad range of concentrations.

[0040] The kit according to the invention comprises:

[0041] a) a first container containing Reagent 1, said Reagent 1consisting of HEPES, EDTA, acetyl-Coenzyme A and DTNB;

[0042] b) a second container containing Reagent 2, said Reagent 2consisting of carnitine acetyl transferase.

[0043] Therefore, one subject of the invention described herein is a kitfor the determination of L-carnitine in biological tissues and fluids,its use for the determination of L-carnitine in biological tissues andfluids and methods for the determination of L-carnitine in biologicaltissues and fluids using the kit.

[0044] According to the invention described herein, the kit permits thedetermination of L-carnitine in biological fluids and tissues. Examplesof biological fluids and tissues are blood, plasma and seminal fluid.When suitably treated according to conventional techniques, othertissues can be analyzed with the kit according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0045] In a first preferred form, Reagent 1 consists of: HEPES 0.1 M, pH7.5 EDTA 50 mM, pH 7 acetyl-Coenzyme A DTNB 10, 1 mM

[0046] Preferably, Reagent 1 has the following composition per ml: HEPES0.1 M, pH 75 0.652 mL EDTA 50 mM, pH 7 0.174 mL acetyl-Coenzyme A 0.087mL DTNB 10.1 mM 0.087 mL

[0047] The Reagent 1 container preferably contains 3 mL of Reagent 1.

[0048] In a first preferred form, Reagent 2 consists of:

[0049] carnitine acetyl transferase 0.500 mL

[0050] If deemed appropriate, the kit may also contain other elementssuch as L-carnitine standards, other reactants for the preparation ofsamples, e.g. plasma or seminal fluid. Reagents 1 and 2 may alsoessentially contain, in addition to the basic reactants necessary forthe reactions, i.e. acetyl-Coenzyme A, DTNB and carnitine acetyltransferase, additional substances useful for the analysis, oralternative substances to those contained in the kit capable ofproducing the same effect, e.g. buffers, chelating agents, complexingagents, pH correctors, preservatives, and stabilizing agents. Anychanges or improvements to the kit supplied on the basis of theinvention described herein, which substantially lead to the same resultand afford the same advantages fall within the compass of the inventiondescribed herein.

[0051] The preferred form of the kit is that in which the reagents arein lyophilized form. This form permits prolonged storage of the reagentsin normal ambient conditions, without particular precautions, withobvious advantages from the point of view of the marketing of the kit.The lyophilization is done according to the conventional techniques usedin the field and requires no further description.

[0052] The kit according to a first embodiment of the inventiondescribed herein (3 mL of Reagent 1 and 0.500 mL of Reagent 2) issufficient for approximately 13 determinations, if the manual method isused, or for approximately 60 determinations, if the automatic method isused. Both methods are described in detail here below.

[0053] A first preferred embodiment of the invention described hereinrelates to the determination of carnitine in plasma.

[0054] The invention described herein is based on the reaction thattakes place between L-carnitine and acetyl-CoA in the presence of theenzyme carnitine acetyl transferase (CAT). The Coenzyme A (CoA) producedby this reaction reacts in turn with 5.5°-dithiobis-2-nitrobenzoate(DTNB), releasing thiophenylate ions (TNB−) which absorb at a wavelengthof 412 nm, in the case of the use of a manual spectrophotometer, or at awavelength of 405 nm, in the case of the use of an automatic-typespectrophotometer. The reactions involved are all quantitative, andtherefore the TNB− absorbance value can be used to determine thecarnitine concentration in the sample.

[0055] The invention is illustrated with reference to a preferred formof its embodiment, namely for the determination of carnitine in plasma.It is understood that the principles involved in the invention describedherein are equally applicable to other tissues, which are treatedaccording to their nature, using conventional procedures which are amatter of general knowledge to people with average experience in thefield. The determination method comprises the following steps:

[0056] a) deproteinizing of the plasma sample in an acid milieu;

[0057] b) neutralization of the sample;

[0058] c) incubation.

[0059] It is in step c) that the enzymatic reactions occur using the kitaccording to the invention described herein: CAT

[0060] The amount of TNB− ions forming in the latter reaction isequivalent to that of the L-carnitine initially present in the sample.

[0061] Ambient pH weakly influences the reactions and therefore theresult of the analysis; in fact, at pH above 8.5 CAT is inactivated,whereas at pH values below 7 the dissociation of DTNB is incomplete, andtherefore the L-carnitine values will be underestimated.

[0062] As an alternative to steps a) and b) described above, the samplecan be deproteinized by simple filtration.

EXAMPLE 1

[0063] Preparation of Solutions

[0064] Acetyl-CoA 12.35 mM: solubilize 86 mg of acetyl-CoA in 8.6 mL ofbidistilled water. This solution will be used to prepare Reagent 1.

[0065] Carnitine acetyl-transferase (CAT): dilute the CAT withbidistilled water (1 volume of CAT+11 volumes of bidistilled water).This solution will be used to prepare Reagent 2. If not usedimmediately, the reagent can be stored at −20° C. and defrosted at thetime of use.

[0066] EDTA 50 mM pH 7.0: solubilize 2.2 g of EDTA in 50 mL ofbidistilled water in a 100 mL calibrated flask. Bring the pH to 7.0 withglacial acetic acid and top up to volume with bidistilled water. Thesolution is stable for 6 months at 4° C.

[0067] DTNB 10 mM: solubilize 34.4 mg of DTNB with 8.6 ml of Hepes 0.1 Mat pH 7.5.

[0068] Hepes 0.1 M at pH 7.5: solubilize 2.38 g of Hepes in 50 mL ofbidistilled water in a 100 mL calibrated flask. Bring the pH to 7.5 withpotash 1.25 M, and top up to volume with bidistilled water. The solutionis stable for 6 months at 4° C.

[0069] Perchloric acid 2.75 M: dilute 23.2 mL of 70% perchloric acid inbidistilled water in a 100 mL calibrated flask. The solution is stablefor 6 months at ambient temperature.

[0070] Potassium carbonate 1.2 M: solubilize 16.6 g of potassiumcarbonate with 80 mL of bidistilled water. Mix and top up to volume withbidistilled water. The solution is stable for 6 months at ambienttemperature.

[0071] Potassium hydroxide 1.25 M: solubilize 8.25 g of potassiumhydroxide with 80 mL of bidistilled water a 100 mL calibrated flask. Mixand top up to volume with bidistilled water. The solution is stable for6 months at ambient temperature.

[0072] Reagent 1: prepare 100 mL of reagent: to 65.5 mL of Hepes 0.1 Mat pH 7.5 add 17.3 mL of EDTA 50 mM, pH 7.0, 8.6 mL of DTNB 10 mM and8.6 mL of acetyl-CoA 12.35 mM. The solution is stable for 6 months at−20° C.

[0073] Deproteinizing of Plasma/Serum and Neutralization of Sample

[0074] Transfer 300 μL of plasma/serum and 40 μL of perchloric acid 2.75M into a 1.5 ml Eppendorf tube. Mix well and then centrifuge at 10,000×gfor 5 min at 4° C. Transfer 250 μL of the supernatant and add 40 μL ofpotassium carbonate 1.2 M in a 1.5 ml Eppendorf tube. Mix and then leavethe samples to digest in ice for 10 min. Centrifuge again at 10,000×gfor 5 min at 4° C. Remove the supernatant for analysis.

[0075] Deproteinizing of Plasma/Serum by Filtration

[0076] Load the sample of plasma/serum on AMICON type filters withcut-off ranging from 5000 to 20000 NMW. Load the sample (400 μL approx.)in an Eppendorf tube containing the filter, to be centrifuged at 15000×gfor 30-40 minutes at 4° C. Remove the filtrate for analysis.

[0077] Enzymatic Analysis

[0078] A) Manual type spectrophotometry.

[0079] Varian DMS-80 UV/VIS spectrophotometer

[0080] Wavelength: 412 nm

[0081] Cuvette: single-use, disposable, 1 cm optical path

[0082] Temperature: 20-25° C.

[0083] Measurement: against air.

[0084] Incubation:

[0085] transfer to cuvette Blank (μL) Sample (μL) Bidistilled water 1000750 Sample — 250 Reagent 1  230 230

[0086]  Stir. After 5 minutes measure the absorbance of the blank andthe sample (A₁). On completion of reading, add 20 μL of CAT (Reagent 2)to each test tube and mix. After another 10 minutes repeat themeasurement of the blank and the sample (A₂).

[0087] The concentration is calculated as follows:$C = {\frac{\Delta \quad A}{ɛ \times d} \times F \times 1000}$

[0088] where:

[0089] C=concentration of sample (nmol/mL)

[0090] ΔA=(A₂−A₁)_(sample)−(A₂−A₁)_(blank)

[0091] ε=coefficient of molar extinction of TNB−(412 nm=13.6L×μmol⁻¹×cm)

[0092] d=optical path length (cm)

[0093] F=dilution factor (6.55)

[0094] 1000=conversion factor to obtain the result in nmol/mL.

[0095] Example of calculation: A₁ A₂ A₂-A₁ Blank 0.169 0.172 0.003Sample 0.170 0.242 0.072

[0096] Applying the formula indicated above: C=33.2 nmol/mL.

[0097] B) Automatic Type Spectrophotometer

[0098] Before starting the analysis, dilute Reagent 1 and Reagent 2 5times with bidistilled water (e.g., to 1 mL of Reagent 1 add 4 mL ofbidistilled water; to 1 mL of Reagent 2 add 4 mL of bidistilled water).

[0099] Experimental Conditions

[0100] Procedure for programming the Roche mod. Cobas-Mira S automaticanalyzer (software used N* 8347) FREE CARNITINE SW 884*7 COBAS MIRA SGENERAL MEASUREMENT MODE ABSORB REACTION MODE D-R-S-SRI CALIBRATION MODELIN REGR REAGENT BLANK REAG/SOL CLEANER NO WAVELENGTH 405 nm DECIMALPOSITION  2 UNIT μmol/L ANALYSIS DILUTION NAME H₂O FACTOR NO TIME NOSTD: MAIN DIRECT MAIN STD 200 μmol/L POST DIL FACTOR  3 CONC. FACTOR NOSAMPLE CYCLE  1 VOLUME  50 μL DILUTION NAME H₂O VOLUME  10 μL REAGENTCYCLE  1 VOLUME 250 μL START R1 CYCLE  7 VOLUME  40 μL DILUTION NAME H₂OVOLUME  10 μL CALCULATION SAMPLE LIMIT  0.1 POINT T1 REAC. DIRECTIONINCREASE CHECK ON CONVERS. FACTOR  1 OFFSET  0 TEST RANGE LOW ON HIGH ONNORMAL RANGE LOW YES HIGH NO NUMBERS OF STEPS  1 CALC. STEP A ENDPOINTFIRST READING  6 LAST READING 30 CALIBRATION CALIB. INTERVAL ON REQUESTBLANK REAGENT RANGE LOW −0.0001 HIGH  0.1000 BLANK RANGE LOW −0.0050HIGH  0.0050 STANDARD POS:  1 1: 200 μmol/L 2: 100 μmol/L 3:  75 μmol/L4:  50 μmol/L 5:  30 μmol/L 6:  20 μmol/L 7:  10 μmol/L 8:  0 μmol/LREPLICATE DUPLICATE DEVIATION 10% CORRECTION STD NO CONTROL CS1* POS: 11LOW 90 mol/L ASSIGN 100 μmol/L HIGH 110 μmol/L CS2* POS: 12 LOW 40μmol/L ASSIGN 50 μmol/L HIGH 60 μmol/L CS3* POS: 13 LOW 12 μmol/L ASSIGN20 μmol/L HIGH 28 μmol/L

[0101] Note: the samples called CS1, CS2 e CS3 refer to the referencestandards and are prepared by scalar dilution of the mother solution ofL-carnitine inner salt at a concentration of 200 μmol/L.

[0102] The calculation of the concentrations of the blanks is doneautomatically by the analyzer which extrapolates them from a straightcalibration line to be prepared fresh for each analysis session(possibly in duplicate).

[0103] The above-described sample preparation and analysis conditionsbeing equal, it is possible to calculate the carnitine concentration foran unknown sample immediately by multiplying its absorbance value by thefactor 1028.382. In this case neither the calibration line nor thecontrol samples will be used.

[0104] Whenever the unknown samples have been deproteinized byacidification and subsequent neutralization with the procedure describedabove, the value of the concentration obtained must be multiplied by thefactor 1.3146.

[0105] Given here below, by way of an example, is a table directlycorrelating the L-carnitine concentrations with the absorbance readings.CALCULATION OF L-CARNITINE CONCENTRATIONS USING THE FACTOR 481.62 Absμmoli/L Abs μmoli/L Abs μmoli/L Abs μmoli/L Abs μmoli/L Abs μmoli/L0.001 0.482 0.051 24.563 0.101 48.644 0.151 72.725 0.201 96.806 0.251120.89 0.002 0.963 0.052 25.044 0.102 49.125 0.152 73.206 0.202 97.2870.252 121.37 0.003 1.445 0.053 25.526 0.103 49.607 0.153 73.688 0.20397.769 0.253 121.85 0.004 1.926 0.054 26.007 0.104 50.088 0.154 74.1690.204 98.250 0.254 122.33 0.005 2.408 0.055 26.489 0.105 50.570 0.15574.651 0.205 98.732 0.255 122.81 0.006 2.890 0.056 26.971 0.106 51.0520.156 75.133 0.206 99.214 0.256 123.29 0.007 3.371 0.057 27.452 0.10751.533 0.157 75.614 0.207 99.695 0.257 123.78 0.008 3.853 0.058 27.9340.108 52.015 0.158 76.096 0.208 100.18 0.258 124.26 0.009 4.335 0.05928.416 0.109 52.497 0.159 76.578 0.209 100.66 0.259 124.74 0.010 4.8160.060 28.897 0.110 52.978 0.160 77.059 0.210 101.14 0.260 125.22 0.0115.298 0.061 29.379 0.111 53.460 0.161 77.541 0.211 101.62 0.261 125.700.012 5.779 0.062 29.860 0.112 53.941 0.162 78.022 0.212 102.10 0.262126.18 0.013 6.261 0.063 30.342 0.113 54.423 0.163 78.504 0.213 102.590.263 126.67 0.014 6.743 0.064 30.824 0.114 54.905 0.164 78.986 0.214103.07 0.264 127.15 0.015 7.224 0.065 31.305 0.115 55.386 0.165 79.4670.215 103.55 0.265 127.63 0.016 7.706 0.066 31.787 0.116 55.868 0.16679.949 0.216 104.03 0.266 128.11 0.017 8.188 0.067 32.269 0.117 56.3500.167 80.431 0.217 104.51 0.267 128.59 0.018 8.669 0.068 32.750 0.11856.831 0.168 80.912 0.218 104.99 0.268 129.07 0.019 9.151 0.069 33.2320.119 57.313 0.169 81.394 0.219 105.47 0.269 129.56 0.020 9.632 0.07033.713 0.120 57.794 0.170 81.875 0.220 105.96 0.270 130.04 0.021 10.1140.071 34.195 0.121 58.276 0.171 82.357 0.221 106.44 0.271 130.52 0.02210.596 0.072 34.677 0.122 58.758 0.172 82.839 0.222 106.92 0.272 131.000.023 11.077 0.073 35.158 0.123 59.239 0.173 83.320 0.223 107.40 0.273131.48 0.024 11.559 0.074 35.640 0.124 59.721 0.174 83.802 0.224 107.880.274 131.96 0.025 12.041 0.075 36.122 0.125 60.203 0.175 84.254 0.225108.36 0.275 132.45 0.026 12.522 0.076 36.603 0.126 60.684 0.176 84.7650.226 108.85 0.276 132.93 0.027 13.004 0.077 37.085 0.127 61.166 0.17785.247 0.227 109.33 0.277 133.41 0.028 13.485 0.078 37.566 0.128 61.6470.178 85.728 0.228 109.81 0.278 133.89 0.029 13.967 0.079 38.048 0.12962.129 0.179 86.210 0.229 110.29 0.279 134.37 0.030 14.449 0.080 38.5300.130 62.611 0.180 86.692 0.230 110.77 0.280 134.85 0.031 14.930 0.08139.011 0.131 63.092 0.181 87.173 0.231 111.25 0.281 135.34 0.032 15.4120.082 39.493 0.132 63.574 0.182 87.655 0.232 111.74 0.282 135.82 0.03315.893 0.083 39.974 0.133 64.055 0.183 88.136 0.233 112.22 0.283 136.300.034 16.375 0.084 40.456 0.134 64.537 0.184 88.618 0.234 112.70 0.284136.78 0.035 16.857 0.085 40.938 0.135 65.019 0.185 89.100 0.235 113.180.285 137.26 0.036 17.338 0.086 41.419 0.136 65.500 0.186 89.581 0.236113.66 0.286 137.74 0.037 17.820 0.087 41.901 0.137 65.982 0.187 90.0630.237 114.14 0.287 138.22 0.038 18.302 0.088 42.383 0.138 66.464 0.18890.545 0.238 114.63 0.288 138.71 0.039 18.783 0.089 42.864 0.139 66.9450.189 91.026 0.239 115.11 0.289 139.19 0.040 19.265 0.090 43.346 0.14067.427 0.190 91.508 0.240 115.59 0.290 139.67 0.041 19.746 0.091 43.8270.141 67.908 0.191 91.989 0.241 116.07 0.291 140.15 0.042 20.228 0.09244.309 0.142 68.390 0.192 92.471 0.242 116.55 0.292 140.63 0.043 20.7100.093 44.791 0.143 68.872 0.193 92.953 0.243 117.03 0.293 141.11 0.04421.191 0.094 45.272 0.144 69.353 0.194 93.434 0.244 117.52 0.294 141.600.045 21.673 0.095 45.754 0.145 69.835 0.195 93.916 0.245 118.00 0.295142.08 0.046 22.155 0.096 46.236 0.146 70.317 0.196 94.398 0.246 118.480.296 142.56 0.047 22.636 0.097 46.717 0.147 70.798 0.197 94.879 0.247118.96 13.297 143.04 0.048 23.118 0.098 47.199 0.148 71.280 0.198 95.3610.248 119.44 0.298 143.52 0.049 23.599 0.099 47.680 0.149 71.761 0.19995.842 0.249 119.92 0.299 144.00 0.050 24.081 0.100 48.162 0.150 72.2430.200 96.324 0.250 120.41 0.300 144.49

1. Kit for the determination of L-carnitine in biological fluids andtissues, consisting essentially of: a) a first container containingReagent 1, said Reagent 1 comprising HEPES, EDTA, acetyl-Coenzyme A andDTNB; b) a second container containing Reagent 2, said Reagent 2comprising carnitine acetyl transferase.
 2. Kit according to claim 1, inwhich said Reagent 1 consists essentially of: HEPES 0.1 M, pH 7.5 EDTA50 mM, pH 7 acetyl-Coenzyme A DTNB 10.1 mM


3. Kit according to claim 1, in which the composition of said Reagent 1is as follows: HEPES 0.1 M, pH 75 0.652 mL EDTA 50 mM, pH 7 0.174 mLacetyl-Coenzyme A 0.087 mL DTNB 10.1 mM 0.087 mL


4. Kit according to claim 3, in which said first container contains 3 mlof said Reagent
 1. 5. Kit according to anyone of claims 1 or 3-4, inwhich said container contains 0.500 ml of said Reagent
 2. 6. Kitaccording to anyone of claims 1-5, for the determination of carnitinesin plasma.
 7. Kit according to anyone of claims 1-5, for thedetermination of carnitines in seminal fluid.
 8. Kit according to anyoneof claims 1-5, for the determination of carnitines in urine.
 9. Kitaccording to anyone of claims 1-8, which additionally comprisescontainers containing carnitine standards.
 10. Kit according to any ofthe foregoing claims, in which the reagents are in lyophilized form. 11.Use of the kit according to claims 1-10 for the determination ofL-carnitine in biological fluids and tissues.
 12. Use according to claim11, in which said fluid is selected from the group consisting of plasma,seminal fluid and urine.
 13. Method for the determination of L-carnitinein biological tissues and fluids, characterized in that it uses the kitaccording to claims 1-10.
 14. Method according to claim 13, in whichsaid fluid is selected from the group consisting of plasma, seminalfluid and urine.